One of the breakthroughs in the treatment of leukemia was the discovery that one of the AML subtypes can be induced to undergo terminal differentiation (18). This led to the discovery of all-trans-retinoic acid (ATRA), and the first ATRA-based treatments resulted in complete remission in a large number of cases (26). Consequently, studies aimed at identifying novel approaches that can be employed for differentiation therapy of AML are considered indispensable (18).
3′-5′-cyclic adenosine monophosphate (cAMP) is implicated in apoptosis, adhesion and differentiation. Multiple hematological malignancies are associated with a deficiency in apoptosis, and cAMP, the first described second messenger, is implicated in the induction of apoptosis and cell differentiation(6). A number of researchers have suggested utilization of the cAMP-related pathways to increase programmed cell death in hematological malignancies(10-12). The pro-apoptotic pathway, promoted by cAMP, depends upon PKA and involves an intrinsic mitochondria-dependent mechanism. Multiple (Bcl)-related family proteins that include pro-apoptotic Bax, Bak, Bad, Bim and anti-apoptotic (such as Bcl-2) proteins are implicated in the release of cytochrome c and Smac (second mitochondria-derived activator of caspases/DIABLO (direct IAP-binding protein with low pI). An increased expression of the Bim protein in response to cAMP/PKA-induced apoptosis was required for induction of G1 phase cell cycle arrest and apoptosis(27). However, the signaling pathway leading from the elevation of cAMP to apoptotic cell death is still under active investigation, and the exact therapeutic targets can be cell-type specific and may vary from patient to patient.
The loss of integrin-dependent adhesion is one of the early hallmarks in the development of precancerous lesions. In solid tumors, such as breast or ovarian cancer, integrins provide a signal that maintains cell attachment to the basal membrane, and supports cell polarization. Loss of beta 1 integrin-dependent adhesion results in a perturbation of normal cell morphology, and can play a role in tumor development. Furthermore, mobilization of hematopoietic progenitors/blasts into the peripheral blood, which is dependent on the alpha 4 beta 1 integrin (VLA-4)(9), can correlate with more aggressive disease in certain leukemias(2). Our recent discovery that cyclic nucleotides (cAMP(4), and 3′-5′-cyclic guanosine monophosphate (cGMP)(3)) can actively down regulate VLA-4 integrin ligand-binding affinity, rapidly down-modulate cell adhesion, and possibly, induce mobilization of hematopoietic progenitors suggested a specific role of these cyclic nucleotides in hematological malignancies.
Furthermore, cAMP is shown to play an important role in leukemic cell differentiation. The cell-permeable analog of cAMP induces differentiation in the human promonocytic U937 cell line(23; 24). The cytoplasmic level of cAMP in this cell line was modulated through the H2 histamine receptor, which at the same time can induce VLA-4 integrin de-activation and cell de-adhesion(4). Thus, it is plausible that modulation of the cAMP level, rather than targeting individual proteins downstream of the cAMP signal, can be utilized as a tool for cell differentiation therapy.
cAMP is synthesized by the family of enzymes termed adenylate cyclases (adenylyl cyclases, ACs). Enzyme activity is controlled by two classes of GPCRs: GalphaS-coupled stimulate cyclase activity, and GalphaI-coupled inhibit the enzyme. Next, cAMP can be hydrolyzed by the superfamily of enzymes called 3′,5′-cyclic nucleotide phosphodiesterases (PDEs), which can have different specificities (cAMP vs. cGMP), localization, and regulation. PDEs are accepted targets for treatment of hematological malignancies, and a number of PDE inhibitors are being tested in different model systems (10).
Another underappreciated mechanism is the removal of the cyclic nucleotides from the cytoplasm by the ATP-binding cassette transporter (ABC-transporter) family of proteins. According to the UCSF-FDA TransPortal database only two transporters, MRP4 and MRP5 are implicated in the active removal of cAMP. Moreover, MRP4 is expressed on the plasma membrane of CD34+ cells, exhibits higher binding affinity for cAMP (vs. cGMP), and the expression of MRP4 (but not MRP5) significantly decreases during leukocyte differentiation (14). This suggests that un-differentiated cell phenotypes can have an increased ability to remove cAMP from the cytoplasm. We recognize that other transporters may participate in this process.
Several clinical studies point toward a specific role of cyclic nucleotides in patients with leukemia. In the urine of patients with four types of leukemia (AML, CML, ALL, and CLL) the concentration and urinary excretion of cyclic nucleotides was higher than in healthy volunteers, with the largest difference between acute leukemia patients and control groups (22). In addition, the plasma level of cyclic nucleotides correlated with the stage of the disease, and it was different in patients who attained remission vs. relapsed individuals (17). At the same time, in WBCs, the cAMP concentration in leukemic cells was lower than in normal cells (16; 22). Based on these and other data we proposed that certain leukemic cells have developed a mechanism that actively removes the pro-apoptotic second messenger (cAMP) from cells into the blood, thus protecting the cell from apoptosis. Since cAMP is excreted through the kidneys, this mechanism explains the lower intracellular cAMP content in leukemic cells (cAMP is continuously removed), the higher plasma and urine concentrations, and the correlation between cAMP concentration and disease progression.
Several recent reviews discuss cyclic nucleotide modulation as a possible option for cancer therapy. Because overexpression of PDE isoforms has been described in several cancers, PDE inhibitors are envisioned as a viable option to restore normal nucleotide metabolism (10; 21). Downstream effectors of the cAMP/PKA-induced apoptotic pathway (such as the Bim protein) are also under investigation for targeting in cancer (1). The interest in cAMP and cGMP efflux in the cancer field was stimulated mainly by the fact that cyclic nucleotides represent natural substrates for multidrug resistance proteins (MRPs/ABCCs), implicated in the efflux of anti-cancer drugs, and not usually envisioned as a mechanism for modulating the signaling for cell reprogramming.
The idea that cell “maturation”, resistant to ATRA-induced differentiation, can be promoted by cAMP-elevating agents, or by using cAMP analogs, is nearly twenty years old (20). An increase in cellular cAMP reduces the effective concentration of ATRA required to achieve maturation(19). The role of the cAMP pathway in t(15; 17) APL has been studied for many years. They uncovered cross-talk between arsenic trioxide and cAMP signaling (29), described the rapid increase in cAMP and PKA expression after ATRA treatment (28), and showed the benefits of the ATRA/arsenic trioxide combination for therapy of APL (25). Several recent reports highlight the role of cAMP/PKA-signaling for cell differentiation. The cAMP analog/ATRA combination is shown to improve the differentiation of t(11; 17)(q23;q21) APL cells, the subset carrying PLZF/RARa fusion that poorly responds to ATRA (7). The activation of the two PKA isozymes is required for ATRA-induced maturation of APL cells (13). Thus, our attention to the mechanisms, modulating cAMP levels is well justified. A seemingly surprising result that the treatment of blasts with cAMP-elevating agents protects cell from cytotoxic drugs (5), can be also interpreted according to our hypothesis: the same class of proteins, which is implicated in the cAMP regulation, can also mediate drug resistance.